Isolation, Identification And Gene Cloning Of Hydrolytic Enzyme For Methylparathion Degradative Strain

Microbial degradation is not only the main pattern of degrading organophosphorous pesticides but also one of powerful means to treat pollution in Environment。Methylparathion (MP, also known as methyl-1605) is a kind of high toxicity organophosphate pesticides and used widely at present throughout China. Its major intermediate during methylparathion degrading process isρ-nitrophenol (PNP), which is soluble and middling toxicity. Human health could be threatened potentially because both MP and PNP have phenyl ring。Four strains in this research,Yw12,Yw15,Yw18,Yw28, with potential ability of degradation for organophosphorus pesticides were isolated from the activated sludge polluted by organophosphorus pesticides.Methyl parathion was used as the sole source of Carbon, Phosphate andρ-nitrophenol was used as the sole source of Carbon by Yw12,and it was identified as Agrobacterium.tumefaciens. Methyl parathion andρ-nitrophenol were used as the sole source of carbon by Yw15,Yw18,Yw28, and they were identified as Ochrobacterum sp, The GenBank accession number of the 16S rDNA sequence of Yw12,Yw15,Yw18,Yw28 are DQ468100,DQ468101, DQ468102 and DQ468103, respectively. At the same time, phylogenetic analysis of Yw12,Yw15,Yw18 and Yw28 were carried out.The four strains can degrade 50mg/L of methyl parathion,ρ-nitrophenol rapidly and completely in 0.5h and 8h respectively, as demonstrated with gas chromatography(GC) and spectrophotometer analysis. At the same time,they can also utilise Methamidophos, Phoxim, Chlorpyriphos, Carbofuran, Deltamethrin, Atraizine,as the sole source of Carbon, which indicates that they have the ability to degrade manifold pesticides .Many methods were used for isolating plasmid of the four strains, and only the alkaline isolate a big plasmid which is bigger than the chromosome. It was reported that the plasmid of many wide strains is bigger than its chromosome, so we speculate that there are plasmid in Yw15,Yw18,and Yw 28. whereas, we introduced many methods for curing the plasmid,but failed.The mpd gene of four strains located in chromosome was coloned by PCR. The size s is 998bp, 999bp,998bp,999bp,respectively and the accession number in Genebank is DQ843605,DQ843606,DQ843607,DQ843608 respectively.The sequence result was blast in Genebank nucleic-nucleic Blast,and the four strains mpd gene have 99% homologous with other Methylparathion degradation strains,which illuminates that the mpd gene are very conservative.The whole-cell protein SDS-PAGE showed that the MPD was component expressed in four strains, and the MPD hydrolase may belongs to endoenzyme.This is the first report that Agrobacterium.tumefaciens Yw12 can degrade methyl parathion andρ-nitrophenol till now. and the four strains shows the potential ability to be used in a bioremediation application for the treatment of methylparathion residues and with further research could lead to an alternative route of disposal for use in agriculture or industry.